Method and apparatus for sample analysis on a continuous flow basis

ABSTRACT

NEW AND IMPROVED METHOD AND APPARATUS FOR THE FULLY AUTOMATIC, HIGHLY ACCURATE QUANTITATIVE ANALYSIS OF A SERIES OF SAMPLES WITH REGARD TO A CONSTITUENT THEREOF ON A CONTINUOUS FLOW BASIS THROUGH RATE REACTION DETERMINATION ARE PROVIDED AND ARE OPERABLE THROUGH THE CONTINUOUS MIXING OF SAID SAMPLES AND A SUITABLE REACTANT IN CONSTANT PROPORTION, AND THE FLOW OF THE RESULTANT SAMPLE-REACTANT MIXTURES BY PUMPING ON A CONTINUOUS BASIS THROUGH REACTION PROMOTING MEANS TO PROMOTE THE SAMPLE CONSTITUENT-REACTANT RATE REACTION IN SUCH MANNER THAT DIFFERENT PORTIONS OF EACH OF THE SAMPLE-REACTANT MIXTURES ARE REACTED FOR DIFFERENT TIMES TO EFFECT A DIFFERENTIAL IN A CHARACTERISTIC OF SAID MIXTURES AS DEVELOPED AS A RESULT OF SAID REACTION. FOLLOWING THIS, THE THUSLY REACTED SAMPLE-REACTANT MIXTURE PORTIONS ARE FLOWED THROUGH DETECTOR MEANS FOR DETECTION OF SAID CHARACTERISTIC DIFFERENTIAL AND RESULTANT PROVISION OF AND OUTPUT SIGNAL WHICH IS INDICATIVE OF THE SAMPLE CONSTITUENT QUANTITY OF INTEREST. SUBSTANTIALLY HYPERBOLIC VARIATION IN THE FLOW RATE OF EACH OF SAID SAMPLE-REACTANT MIXTURES THROUGH SAID REACTION PROMOTING MEANS IS UTILIZED TO PROVIDE FOR THE DIFFERENT SAMPLE-REACTANT MIXTURE PORTION REACTION TIMES. AS SPECIFICALLY DISCLOSED HEREIN, THE METHOD AND APPARATUS OF THE INVENTION ARE PARTICULARLY ADAPTABLE TO THE ANALYSIS OF A SERIES OF BLOOD SAMPLES WITH REGARD TO AN ENZYME CONSTITUENT THEREOF.

Oct. 17, 1972 T. SKEGGS METHOD AND APPARATUS FOR SAMPLE ANALYSIS ON A CONTINUOUS FLOW BASIS 3 Sheet s-Sheet 1 Filed Aug. 31, 1970 vm zhwwfimum $6: KW E wZmo n23. ud

INVENTOR EONARD T. SKEGGS BY ATTORNEY Oct. 17, 1972 T. SKEGGS METHOD AND APPARATUS FOR SAMPLE ANALYSIS ON A CONTINUOUS FLOW BASIS C5 Sheets-Sheet 2 Filed Aug. 31, 1970 OMT mmomouwm muhwEEOJOU N TI WK w mm Ill.

Oct. 17, 1972 T. SKEGGS METHOD AND APPARATUS FOR SAMPLE ANALYSIS ON A CONTINUOUS FLOW BASIS 3 Sheets-Sheet 3 Filed Aug. 31, 1970 SAMPLE,WASH AND SAMPLE, WASH AND AIR ASPIRATION AIR ASPIRATION 2 a8 558$: Q2 3.2: 1 535 NEE 65. 92 5150 ii REAL TIME TR ES IO SLOPE SAMPLE 2 ES l0 SLOPE=IM SAMPLE 3 ESI RECORDER BASELINE SAMPLE INCUBATION TIME TI INVENTOR LEONAR T. SKEGGS BY ATTORNEY United States Patent 3,699,004 METHOD AND APPARATUS FOR SAMPLE gSAgYSIS ON A CONTINUOUS FLOW Leonard T. Skeggs, Kirtland, Ohio, assignor to Technicon Instruments Corporation, Tarrytown, N.Y. Filed Aug. 31, 1970, Ser. No. 68,297 Zlnt. Cl. G01n 31/14 US. Cl. 195103.5 R 14 Claims ABSTRACT OF THE DISCLOSURE New and improved method and apparatus for the fully automatic, highly accurate quantitative analysis of a series of samples with regard to a constituent thereof on a continuous fiow basis through rate reaction determination are provided and are operable through the continuous mixing of said samples and a suitable reactant in constant proportion, and the flow of the resultant sample-reactant mixtures by pumping on a continuous basis through reaction promoting means to promote the sample constituent-reactant rate reaction in such manner that different portions of each of the sample-reactant mixtures are reacted for different times to effect a differential in a characteristic of said mixtures as developed as a result of said reaction. Following this, the thusly reacted sample-reactant mixture portions are flowed through detector means for detection of said characteristic differential and resultant provision of and output signal which is indicative of the sample constituent quantity of interest. Substantially hyperbolic variation in the flow rate of each of said sample-reactant mixtures through said reaction promoting means is utilized to provide for the different sample-reactant mixture portion reaction times. As specifically disclosed herein, the method and apparatus of the invention are particularly adaptable to the analysis of a series of blood samples with regard to an enzyme constituent thereof.

BACKGROUND OF THE INVENTION (1) Field of the invention This invention relates to new and improved method and apparatus for the fully automatic, highly accurate quantitative analysis of a series of samples on a continuous flow basis through rate reaction determination and, more particularly, to such method and apparatus as are particularly applicable to the fully automatic, highly accurate quantitative analysis of a series of blood samples on a continuous flow basis with regard to one or more enzyme constituents thereof.

(2) Description of the prior art Although a wide variety of method and apparatus are currently available for the quantitative analysis, for example, of blood samples with regard to one or more enzyme constituents thereof as described, for example, in the booklet Diagnostic Enzymology published by Dade Reagents Inc. of Miami, Fla. in 1966 and the undated booklet The Theory of Enzyme Tests" as published by Boehringer Mannheim Corporation of New York, N.Y., it may be understood that no prior art method and/ or apparatus are known which can provide for the automatic, highly accurate quantitative analysis of a plurality of blood samples with regard to one or more enzyme constituents thereof on a continuous fiow basis.

More specifically, it may be understood that many of the currently utilized methods and apparatus for enzyme determination of the prior art are wholly or partially manual, to thus introduce the most significant disadvantages of human error and inordinately high skilled tech- 3,699,004 Patented Oct. 17, 1972 nician costs to the enzyme determination procedure, and to render the same inapplicable for use on a continuous flow basis. Too many of the prior art methods and apparatus for enzyme determination require precise sample and substrate temperature equilibration prior to the mixing thereof since the same are not automatically brought to the same temperature prior to mixing.

Further, many of the methods and apparatus of the prior art rely, in the determination of the enzyme-substrate rate reaction, on the use of a reference standard which is constituted by an enzyme dissolved in a serum base, and the standard value of which was initially determined by classical rate reaction techniques. As a result, it is believed clear that the accuracy and validity of any enzyme determination which requires the use of such reference standard becomes wholly dependent upon the accuracy and care with which said reference standard was prepared. This is to say that any error in the preparation of said reference standard will, of course, render substantially useless the enzyme determination results of interest. Too, the respective constituents of such reference standards, and the obvious care with which the same must, of necessity, be prepared, can result in a relatively high cost of such reference standards to further disadvantage.

Another problem which arises in enzwne determination through use of the methods and apparatus of the prior art resides in the fact that the same accomplish such determination through the measurement and calibration of two points, only, of the relevant enzyme rate reaction curve, to thereby require the not always valid assumption that the results of such enzyme rate reaction would be linear between said two points. An additional significant disadvantage which is inherent in many of the prior art methods and apparatus for enzyme determination resides in the fact that the same do not always effect the measurement of the enzyme rate reacting under the most optimum conditions which occur at the initial portion of the rate reaction velocity curve, and following which substrate inhibition may occur to inhibit or limit the desired enzymesubstrate forward reaction and thus provide invalid results.

In addition, many of the methods and apparatus of the prior art are found to be significantly lacking in versatility to thus render substantially impractical the change from the determination of one enzyme to the determination of another enzyme.

OBJECTS OF THE INVENTION It is, accordingly, an object of this invention to provide new and improved method and apparatus for the fully automatic, highly accurate quantitative analysis of a series of samples on a continuous flow basis through rate reaction determination.

Another object of this invention is the provision of method and apparatus as above which are particularly adaptable to the fully automatic, highly accurate quantitative analysis of a series of blood samples with regard to one or more enzyme constituents thereof.

Another object of this invention is the provision of method and apparatus as above which automatically provide for precise sample and substrate temperature equilibration prior to the mixing thereof.

Another object of this invention is the provision of method and apparatus as above which eliminate the need for the use of reference standards in such rate reaction determination.

Another object of this invention is the provision of method and apparatus as above which are of relatively simple construction, and which require the use of only readily available components of proven dependability therein to thereby provide for long periods of satisfactory, substantially maintenance-free apparatus operation.

Another object of this invention is the provision of method and apparatus as above which are of significant versatility in that the same are applicable with but minor modifications to the determination of a wide variety of rate reactions.

A further object of this invention is the provision of new and improved method and apparatus as above which are particularly applicable for use in the formation of one or more enzyme analysis channels in automatic blood sample supply, treatment and analysis apparatus of the nature disclosed, for example, in U.S. Pats. 3,134,263 and 3,241,432.

SUMMARY OF THE INVENTION As disclosed herein, the new and improved method and apparatus of the invention are applied by way of example to the automatic quantitative analysis of a series of blood samples with regard to the determination of one or more enzyme constituents of said blood samples on a continuous flow basis through rate reaction determination. More specifically, in the quantitative analysis of a blood sample with regard to an enzyme constituent thereof through rate reaction determination, it may be understood that said blood sample is mixed with a reactant taking the form of a substrate to effect said rate reaction, and that the concentration of said enzyme is arrived at by determining the rate at which the same catalyzes the reaction. This is to say that the concentration of said enzyme is determined through the measurement of its catalytic activity with regard to a particular substrate by the determination of the amount of product formed in a certain amount of time by said rate'reaction.

For such use, the apparatus of the invention comprise operatively associated sample and substrate supply means; sample and substrate supply rate control means; sample and substrate mixing and treatment means which include a mixing and incubation coil; sample and substrate mixture and color producing reagent supply, mixing and treatment means; reaction rate analysis means; and reaction rate analysis results recorder means, respectively.

In operation as generally described, a stream of a series of said samples, and a stream of said substrate are provided from said sample and substrate supply means for merger in fixed proportion with attendant air segmentation of the resultant sample-substrate mixture into a large plurality of sample-substrate mixture portions, and the resultant sample-substrate mixture is supplied to the mixing and incubation coil of said sample and substrate mixing and treatment means for appropriate incubation and promotion of the rate reaction of interest. 'Following this, the thusly incubated sample-substrate mixture is supplied to said reaction rate analysis means which take the form of a colorimeter and are operable to detect the changes in optical density of said samplesubstrate mixture occasioned by said rate reaction and to provide an output which is indicative thereof to operate said recorder means.

For use in accordance with the teachings of this invention which dictate a substantially hyperbolic variation in the flow rate of the respective sample-substrate mixture portions through the mixing and incubation coil for each of said samples, it may be understood that said sample and substrate supply rate control means will, for example, be operative to commence the substantially hyperbolic reduction of said flow rate from a fast value thereof to a slow value thereof as the leading portion of a sample-substrate mixture commences entry into said mixing and incubation coil, and will be effective to complete said hyperbolic flow rate reduction substantially concomitantly with the completion of the flow of the trailing portion of said sample-substrate mixture from said mixing and incubation coil. Thus may be understood whereby the extent to which said rate reaction is enabled to proceed for different portions of said sample-substrate mixture will be varied substantially linearly with attendant variation in the change in the optical densities of said sample-substrate mixture portions. When utilized in this manner, it may be understood that the curve provided by the recorder means in response to the operation as described of the colorimetric analysis means will be constituted by the sample-substrate mixture optical density developed as a result of the enzyme-substrate reaction as plotted against sample incubation time, whereby the slope of this curve will be directly indicative, without conversion, of the amount of the enzyme of interest present in the blood samples.

For use in enzyme determination through colorimetric analysis in those instances wherein the provided enzymesubstrate rate reaction will not produce a satisfactorily detectable change in the optical density or color of the sample-substrate mixture to enable colorimetric analysis, it may be understood that the mixed and incubated sample-substrate mixture will be sampled at substantially constant flow rate by said fluid sample and substrate mixture and color producing reagent supply, mixing and treatment means for mixture in substantially constant, predetermined proportions with a suitable color producing reagent and subsequent flow through said colorimetric analysis means for provision of the analysis results as described.

The above and other objects and significant advantages of this invention are believed made clear by the following detailed description thereof taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a combined schematic and flow diagram of new and improved apparatus constructed and operative in accordance with the teachings of this invention;

FIG. 2 is a combined schematic and flow diagram of a portion of apparatus of the nature of those depicted in FIG. 1 but which include a secondary reaction circuit;

FIG. 3 illustrates fluid flow through the mixing and incubation coil of the apparatus of FIG. 1;

FIG. 4 is a graph of pump output and flow rate through the mixing and incubation coil of the apparatus of FIGS. 1 and 2 as plotted against real time for substantially hyperbolic change in said pump output and flow rate; and

FIG. 5 is a graph of the sample-substrate mixture portions optical densities developed as a result of the enzyme-substrate reaction as plotted against sample-substrate mixture portion incubation times by the recorder of the apparatus of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION Referring now to FIG. 1, new and improved apparatus constructed and operative in accordance with the teachings of this invention for use in the fully automatic, highly accurate quantitative analysis of a series of fluid samples on a continuous flow basis with regard to one or more enzyme constituents thereof are indicated generally at 20, and comprise fluid sample and substrate supply means as indicated generally at 22, fluid sample and substrate supply rate control means as indicated generally at 24, fluid sample and substrate mixing and treatment means as indicated generally at 26, reaction rate detection means as indicated generally at 30, and reaction rate analysis results recorder means as indicated generally at 32, respectively.

The fluid sample and substrate supply means 22 may, for example, take the general form of those shown and described in U.S. Pat. 3,134,263, issued May 26, 1964 to Edward B. M. DeJong and assigned to the assignee hereof, and comprise a turntable 34 upon' which is disposed a generally circular array of blood sample containers 36. A sample olT-take device is indicated at 38 and comprises a sample off-take probe 40 and probe operating means 42, respectively. A wash liquid receptacle 44 is disposed as shown adjacent the turntable 34, while a sample drive motor is indicated at 46 and is operatively connected as indicated by the dashed lines to drive each of the turntable 34 and the sample off-take probe operating means 42 in synchronized manner under the control of a timer 48 which is electrically connected as indicated by line 50 to said sample drive motor.

A proportioning pump which may, for example, take the general peristaltic pump form of that shown and described in U.S. Pat. 3,227,091, issued Jan. 4, 1966 to Jack Isreeli and assigned to the assignee hereof is indicated in dashed lines at 52, and comprises compressible pump tubes 54, 56, and 58 which are progressively compressible or occludable in perfect phase relationship in the direction from left to right as seen in FIG. 1 by non-illustrated pump rollers to pump fluids therethrough. A DC pump drive motor is indicated at 60 and is, of course, operable to drive the pump 52 with the rate of fluid flow through each of the compressible pump tubes 54, 56 and 58 being determined by the respective internal diameters thereof and the rate at which said pump is driven by the DC pump drive motor 60. The outlet end of the sample off-take probe 40 is connected as indicated to the inlet end of the compressible pump tube 54, while the inlet end of the compressible pump tube 56 extends as shown into a substrate container 62 to pump substrate from the latter. The inlet end of compressible pump tube 58 is left open as shown to atmosphere to pump air therethrough.

The fluid sample and substrate supply rate control means 24 comprise an operational amplifier 64 having a feedback resistor 66 connected thereacross in line 67 in conventional manner, and a potentiometer 68, having a variable resistance RV, connected as shown in line 70 at the input side of said operational amplifier. The output of the operational amplifier 64 is applied as indicated on line 71 to energize the DC pump drive motor 60 to drive the latter. The timer 48 is operatively connected as indicated by the dashed line to the potentiometer 68 to enable the precise control of the variable resistance RV, and accordingly of the speed of the DC pump drive motor 60, at any point in time from said timer. More specifically, it may be understood that the speed of the DC pump drive motor 60 at any point in time may be determined in accordance with Equation 1 as follows:

Equation 1 RF Vm-JcE RV wherein; Vm equals the speed of rotation of DC pump drive motor 60, k equals a constant, B equals the input voltage applied as indicated to line 70, RF equals the resistance of feedback resistor 66 and RV equals the resistance of the potentiometer 68.

The speed of the pump drive motor 60 and accordingly, the respective sample and substrate supply rates provided by the pump 52, may respectively be precisely determined and varied as controlled by the timer 48.

The fluid sample and substrate mixing and treatment means 26 comprise a substrate pre-heating or pre-incubation coil 72 which is disposed as indicated in suitable temperature control means in the nature, for example, of a temperature control bath 74 which is effective to maintain the temperature of said coil substantially at a desired level. A junction conduit is indicated at 76 and the junction conduit inlets are connected as shown to the respective outlet ends of compressible pump tubes 56 and 58, while the outlet of the junction conduit 76 is connected as shown by a connecting conduit 77 to the inlet of the substrate pre-heating or pre-incubation coil 72.

A substrate and sample mixture incubation coil is indicated at 78 and is also disposed as shown in the temperature control bath 74 which is of course effective to maintain the temperature of the said incubation coil substantially at the same desired level as the pre-heating or pre-incubation coil 72. In addition, a substantial portion of the sample stream inlet of the junction conduit 82 is also disposed as shown in the temperature control bath 74 for obvious purpose. A junction conduit is indicated at '82 and the respective outlets of the compressible pump tube 54 and the substrate pre-incubation coil 72 are connected as shown to the inlets of the junction conduit 82, while the outlet of said junction conduit is connected to the inlet of the substrate and sample mixture coil 78. As a result of this arrangement it is believed made clear that substantially precise sample and substrate temperature equilibration is automatically achieved to significant advantage in that the same are automatically brought to substantialy the same temperature prior to the merger thereof in junction conduit 82.

The reaction rate detection means 30 comprise a colorimeter as indicated at 124 which, of course, includes a non-illustrated colorimeter flow cell, and which will probably include non-illustrated debubbler means disposed upstream of said flow cell to effect the substantial removal to atmosphere of any gas segments in the fluid stream flowing therethrough. A conduit 96 is provided to connect the outlet of the incubation coil 78 to the inlet of said non-illustrated colorimeter flow cell to flow fluids therethrough for enzyme rate reaction detection purposes in accordance with the optical density of said fluids as determined by said colorimeter means in conventional manner. A colorimeter outlet conduit 128 is provided to flow fluids to waste after the passage thereof through said colorimeter means.

The reaction rate analysis results recording means 32 comprise a DC null-balance type strip chart recorder 130 having a strip chart 132 which is driven in the indicated direction, and a recording pen or stylus 134 operatively associated with said strip chart and operable to trace a curve 136 on the latter to provide a permanent and readily reproducible record of the enzyme rate reaction analyses of interest as described in greater detail hereinbelow.

The form of the apparatus of the invention for use in those instances wherein the enzyme-substrate rate reaction will not produce a satisfactorily detectable change in the optical density of the sample-substrate mixture to enable colorimetric analysis of the latter is indicated generally at 97 in FIG. 2 and may be seen to include secondary, sample-substrate mixture and color producing reagent supply, mixing and treatment means as indicated generally at 28 and which are operatively disposed as shown intermediate the incubation coil 78 and the colorimeter 124.

The secondary circuit means 98 comprise a proportioning pump as indicated in dashed lines at 84 and which may, for example, take the same peristaltic pump form as the pump 52. The proportioning pump 84 is driven at constant speed by an AC pump drive motor 86, and comprises compressible pump tubes 86, 88 and 90 which are again respectively compressible or occludable in perfect phase relationship by non-illustrated pump rollers in the direction from left to right to pump fluids therethrough in said direction.

A junction conduit is indicated at 9'8 and the inlet thereof is connected as shown to the outlet of the conduit 96 to flow the sample-substrate mixture stream to said junction conduit. One outlet of the junction conduit 98 extends as shown to waste, while the other outlet thereof is connected as shown to the inlet of compressible pump tube 86. The inlet of compressible pump tube 88 extends as shown into a container 92 of a suitable color producing reagent, while the inlet of compressible pump tube 90 is left open to atmosphere.

A junction conduit is indicated at 104 and functions to merge the respective reagent and air outputs of pump tubes 88 and 90 to thus air-segment the reagent stream provided by the former. This air-segmented reagent stream is merged as shown in a junction conduit 106 with the sample-substrate stream from compressible pump tube 86.

A mixing coil is indicated at 108 and the outlet of junction conduit 106 is connected as shown to the inlet of said mixing coil. Temperature control means which may again, for example, take the form of a temperature control bath are indicated at 110 and are effective to maintain the temperature of the mixing coil 108 at a desired level.

A conduit 109 is provided to connect the outlet of mixing coil 108 to the inlet of the colorimeter 124 for the flow of the suitably mixed and reacted, air-segmented sample-substrate-reagent stream to the latter. If required, dialyzer means (not shown) may of course be provided in the secondary circuit means 28.

OPERATION In operation as generally described for the quantitative analysis of a series of samples with regard to a constituent thereof through the mixture on a continuous flow basis of each of said samples with an appropriate reactant to effect a constituent-reactant rate reaction with attendant change in a detectable characteristic of the resultant sample-reactant mixture, it may be understood that the extent to which said rate reaction is enabled to proceed is varied for different portions of said samplereactant mixture to generate a detectable differential in said mixture, which differential is indicative of the rate of said reaction, and accordingly of the quantity of said constituent, and that said characteristic differential is subsequently detected to provide the desired results.

More specifically, and for use, for example, in the quantitative analysis of a plurality of blood samples to determine the amount of the enzyme constituent alkaline phosphatase present in each of said blood samples, through the reaction thereof with an appropriate reactant taking the form of a substrate in the nature of synthetic substrate para-nitrophenylphosphate, by the mixture of said enzyme and substrate, and the measurement of the rate reaction therebetween at a large plurality of different incubation times during said reaction, it may be understood that each of the sample containers 30 on the turntable 34 would be filled with a blood serum sample from a different patient, While the substrate container 62 would be filled with said synthetic substrate. It is believed clearly understood by those skilled in this art that the use of the secondary supply, mixing and treatment means 28 would not be required for the quantitative analysis of blood samples with regard to the enzyme alkaline phosphatase. The temperature control means 74 would be adjusted, if necessary, to maintain the respective temperature of the substrate pre-incubation coil 72 and the substrate and sample mixture incubation coil 78 at substantially 37 C. Too, the timer 48 and potentiometer 68 would be adjusted to provide for the continuous cycling of the speed of rotation Vm of the DC pump drive motor 60 in substantially hyperbolic manner between a first or fast speed V2 and a second or slow speed V1 for each sample-substrate mixture as described in much greater detail hereinbelow.

With the apparatus arranged as described, the turntable 34 is intermittently rotated, o-r indexed, to present each of the blood serum sample containers 36 in turn to the sample off-take probe 40, while the latter is in turn intermittently operated to immerse the inlet end of said probe in a thusly presented sample container for a predetermined period of time to aspirate, through compressible pump tube 54, a predetermined volume of the blood serum samples from said container, to then transfer said off-take probe inlet end through the ambient air for immersion in the wash liquid receptacle 44 for a predetermined period of time to thus aspirate a predetermined volume of ambient air for use as a separating and cleansing fluid, followed by a predetermined volume of said Wash liquid therethrough for similar purpose, and to again transfer said off-take probe inlet end through the ambient air for immersion in the next presented sample container 36 for a predetermined period of time, to thus aspirate another predetermined volume of ambient air therethrough and commence the aspiration of a like, predetermined volume of the blood serum sample from said next presented sample container.

As a result, it may be understood that a fluid stream consisting of successive, predetermined volumes of said blood serum samples as spaced, in each instance, by a segment of air, a slug of said wash liquid, and a segment of air, respectively, will be supplied from the sample olftake probe 40 through compressible pump tube 54 tothe relevant inlet of junction conduit 82.

Concomitantly, a stream of the substrate will be aspirated from the substrate container 62 through compressible pump tube 56, and a stream of ambient air will be aspirated through the compressible pump tube 58 for merger as indicated with said substrate stream at the r' junction conduit 76 with resultant air segmentation of said substrate stream and supply thereof through connecting conduit 77 to the substrate preincubation coil 72. Flow of this air-segmented substrate stream through said preincubation coil will, of course, result in suitable substrate pre-incubation to the desired extent as determined of course by the coil temperature, the substrate flow rate through said pre-incubation coil, and the effective length of the latter. Following this, the thusly air-segmented and pre-incubated substrate stream will flow as indicated to the other inlet of junction conduit 82 for merger therewithin with the sample stream flowing thereto as described from compressible pump tube 54.

As a result of this merger of the sample and substrate streams, and referring now to FIG. 3 which depicts the incubation coil 78 in straight-line manner for purposes of illustration, it may be understood that a sample-substrate mixture stream S consisting of successive ones of said blood samples as mixed with said substrate, and as preceded and followed by air, wash liquid and air segments, respectively, will be flowed from the junction conduit 82 to and through the substrate and sample mixture incubation coil 7 8.

Assuming for example that each of said sample-sub strate mixtures is divided or air-segmented into ten samplesubstrate mixture volumes or slugs and that the total volume of a thusly air-segmented sample-substrate mixture is approximately 25% greater than the total volume of the incubation coil 78, it may be understood that FIG. 3 depicts by way of example the apparatus 20 substantially at the point in time wherein the leading samplesubstrate mixture segment ES1 of sample 3 has completed its flow through the mixing and incubation coil 78.

As discussed, the timer 48 would be arranged to substantially continuously vary the resistance RV of the potentiometer 68 between upper and lower limits or velocities V2 and V1 during each blood sample analysis operating cycle to effect a substantially hyperbolic change in the velocity of the DC pump drive motor 60 and accordingly in the pumping rate through compressible pump tubes 54, 56 and 58 as provided by proportioning pump 52.

For use in the automatic, successive colorimetric analysis of a plurality of blood samples with regard to the quantity of the enzyme alkaline phosphatase present in each of said blood samples through use of the synthetic substrate para-nitrophenylphosphate as described, and referrmg again to FIG. 3, it may be understood that the pump drive motor 60 would be operated as described at the fast or V2 velocity thereof until the first samplesubstrate segment ESl of sample 3 had commenced to flow through the mixing and incubation coil 78, whereupon the speed of said pump drive motor would commence to be substantially hyperbolically shifted or reduced to arrive at the slow or V1 velocity thereof substantially concomitantly with the completion of the flow of all of the sample-substrate mixture segments BS1 through E510 of sample 3 through the coil 78.

As this occurs, the DC pump drive motor 60 would be substantially instantaneously returned to the fast or V2 velocity thereof and maintained at said fast or V2 velocity to minimize succeeding blood sample, wash liquid and inter sample air segment aspiration times as described. Following this, as the leading edge of the first samplesubstrate mixture volume or segment ESl of sample 4 arrives at the entrance end of the incubation coil 78 to commence its flow therethrough, the hyperbolic reduction in the velocity of said pump drive motor to the slow or V1 velocity thereof would again be commenced.

As a result of this substantially hyperbolic change in pump drive motor velocity, and accordingly of the flow rate of the sample-substrate mixture segments for sample 3 through the incubation coil 78, it may be understood that the first sample-substrate mixture segment ESl of sample 3 will have spent the least amount of time in flowing through the incubation coil 78, while the last samplesubstrate mixture segment E810 of sample 3 will have spent the greatest amount of time in flowing through the incubation coil 78. Each of the intervening sample-substrate mixture segments ES2 through 'ES9 of sample 3 will have spent successively greater amounts of time in flowing through the incubation coil 78 as determined by the proximity of said segments to the segment E510. Thus, each of the sample-substrate mixture portions BS1 through E810 in that order will spend a substantially linearly increasing amount of time in the incubation coil 78. As a result it is believed clear that the extent to which the enzyme-substrate rate reaction is enabled to proceed in said incubation coil for each of said segments will likewise be increased substantially linearly to thus provide for an increase in the respective optical densities of each of the sample-substrate mixture portions ESl through ES which are developed as a result of the enzyme-substrate rate reactions of interest. An optical density dilferential will accordingly be provided, and such differential may readily be utilized as described in detail hereinbelow to arrive at the enzyme quantity results of interest.

Referring now to FIG. 4 for more detailed illustration of the effect of this hyperbolic speed control upon samplesubstrate mixture flow rate, curve 154 illustrates the pump output and flow rate through mixing and incubation coil 78 as plotted against real time TR, and it may be seen that said flow rate will be maintained at the value thereof which corresponds to the fast or V2 pump drive motor velocity for blood sample, wash and air aspiration, will commence the hyperbolic variation thereof substantially coincident in point of time with the entry of the samplesubstrate mixture volume BS1 into the incubation coil 78, will be varied by hyperbolic reduction down to the value thereof which corresponds to the slow or V1 velocity of said pump drive motor concomitant with the flow of sample 3 through said mixing coil, will again be shifted substantially instantaneously to the value thereof which corresponds to said fast or V2 velocity of said pump drive motor for succeeding blood sample, Wash and air segments aspiration, and will again be reduced by hyperbolic variation to the lower value thereof concomitant with the flow of sample 4 through said mixing and incubation coil.

Upon exit from the incubation coil 78 the samplesubstrate stream S will flow through conduit 96 to the colorimeter means 124 with the non-illustrated debubbler means of the latter functioning, if included, to remove the respective inter sample and intra sample air segments A therefrom, whereupon the now substantially air segment-free stream will flow through the non-illustrated colorimeter flow cell for colorimetric analysis thereof, and therefrom to waste as indicated through conduit 128.

FIG. 5 depicts the curve 136 which will be provided on the strip chart 132 of the recorder 130 in response to hyperbolic operation as described of the apparatus 20. Curve 136 may, in this instance, be understood to be directly representative of the sample-substrate mixture portions optical densities developed as a result of the sample enzyme-substrate reaction, as plotted against sample incubation time TI, whereby the slope M of the curve 136 of FIG. 4 may be read directly and utilized 10 without conversion to arrive at the desired determination of the milli international units of enzyme per milliliter of blood sample.

More specifically, it may be understood that the actual quantitative determination of the enzyme of interest in blood sample 3 under discussion may now be readily and precisely effected through use of the following standard equation:

Equation 3 Ma 6 H m1 MX10 X X 6 wherein:

mIu m1 equals the milli international units of enzyme per milliliter of blood sample, M equals the slope of curve 136 of FIG. 8 as provided by the recorder 130, TV equals the total sample and substrate volume, SV equals the sample volume which when divided into TV gives the dilution factor, and 1 equals the molar extinction factor as determined for the apparatus 20.

For use as described with a total available apparatus cycle time per blood sample analysis of approximately three minutes, of which seconds are available for blood sample and substrate mixing and incubation, and the remaining 20 seconds of which are utilized for blood sample, wash liquid and inter sample air segment aspiration, it may be understood that the pump drive motor 60 may be operated at the fast or V2 velocity thereof for approximately 20 of these seconds to provide for an approximately 20 second period for blood sample, wash liquid and inter sample air segment aspiration, and may be operated to provide for a maximum sample-substrate mixture portion incubation time of 160 seconds for the mixture portion E510. Too, a preferable ratio between V2 and V1 velocities of the pump drive motor 60 may, for example, be approximately 6:1. In addition, for analysis as described of the blood sample with regard to the enzyme alkaline phosphatase through use of the synthetic substrate paranitrophenylphosphate, it may be understood that the respective internal diameters of the compressible pump tubes 54 and 56 might be arranged to provide for a substrate to enzyme ratio of approximately 20:1.

For use of the apparatus of the invention in the quantitative analysis of a plurality of blood samples with regard to an enzyme in the nature of creatine phosphokinase, or CPK, through the reaction thereof with a substrate in the nature of creatine phosphate, it will be understood by those skilled in this art that use of the ap paratus 97 of FIG. 2, which include the secondary, sample and substrate mixture and color producing reagent supply, mixing and treatment means 28 to add a suitable color producing reagent to the incubated and reacted sample-substrate mixture to provide for a readily detectable change in the optical density thereof, will become necessary. More specifically, and for such use, it may be understood that the pump drive motor 86 would be operated to drive proportioning pump 84 at constant speed. For quantitative analysis as described with regard to the enzyme CPK, the color producing reagent container 92 would be filled with an appropriate color producing reagent in the nature, for example of diacetyl orcinol.

In operation, the respective fluid sample and substrate supply means 22, fluid sample and substrate supply rate control means 24, fluid sample and substrate mixing and treatment means 26, reaction rate detection means 30, and reaction rate analysis results recorder means 32 would be operated substantially as described hereinabove with the described results insofar as operation to the downstream end of the incubation 78 is concerned. Under these conditions, however, as the suitably incubated sample-substrate mixture stream S flows into junction conduit 98 through conduit 96, the same would in essence be sampled at substantially constant flow rate through the relevant outlet of said junction conduit and compressible pump tube 86 while the excess thereof, if any, would simply be flowed to waste as indicated through the other outlet of junction conduit 98; it being understood that the substantially constant pumping rate through compressible pump tube 86 would preferably be arranged through appropriate choice of compressible pump tube internal diameter and pump velocity to insure that said pumping rate would, under all conditions, be less than the flow rate through conduit 96.

concomitantly, the color producing reagent would be pumped at substantially constant flow rate from container 92 to junction conduit 104 and air-segmented in the latter by the ambient air pumped thereto at substantially constant flow rate through compressible pump tube 90, with the resultant air-segmented color producing reagent stream being merged as indicated with the sample-substrate mixture stream S in the junction conduit 106. Following this, the resultant sample-substrate-reagent mixture stream would be flowed to and through the coil 108 for thorough mixing and heating and/ or simple time delay, depending upon the nature of the desired reaction as discussed hereinabove.

Upon exit from the coil 108, the now suitably incubated and reacted sample-substrate-reagent mixture stream would be flowed through conduit 109 to and through the non-illustrated flow cell of the colorimeter 124 as described for automatic, successive colorimetric analysis of the respective blood samples and attendant provision of the readily interpretable and reproducible curve 136 on the strip chart 134 of the recorder 130 as described.

Although disclosed hereinabove by way of illustrative example, only, as directed to the colorimetric quantitative analysis of a plurality of blood serum samples with regard to a particular enzyme constituent thereof, it is believed clear that the new and improved method and ap paratus of this invention would be equally applicable for use in such enzyme determination with regard to fluid samples other and different than blood serum samples. Too, it is believed equally clear that the new and improved method and apparatus of the invention would also be particularly applicable to the determination as described of rate reactions between substances other and different than enzymes and substrates, respectively, for quantitative analysis of samples which may or may not be blood samples with regard to said other and dilferent substances.

Although disclosed by way of example as operating in fast to slow manner attendant the substantially hyperbolic variation in the pump drive motor speed concomitant with the flow of the sample-substrate mixture for each of said blood samples through the mixing and incubation coil 78, it is believed clear that said apparatus can, alternatively, to operated in slow to fast manner attendant such hyperbolic variation. Further, although disclosed for purpose of example as a single channel device, it may be understood that the apparatus of the invention may, of course, be operated as a multi-channel device to substantially increase the rate of analyses performed thereby per hour. Thus, for example, the turntable 34 might include four generally concentric, circular arrays of sample containers, and four simultaneously operable sample olf-take means 38 might be provided with similar duplication in compressible pump tubes, pre-incubation and mixing and incubation coils, connecting conduits, colorimeter flow cells, and strip chart recorder means to enable the substantially, concomitant enzyme analysis of four blood samples per apparatus cycle. As a further alternative, the apparatus 20 might be arranged and operated to effect the colorimetric quantitative analysis of, for example, each of a plurality of blood samples with regard to four different enzyme constituents thereof and such could, of course, be readily effected through the division of the sample as aspirated through the sample off-take probe into four portions and the provision of four substantially concomitant mixing, incubation, and colorimetric analysis of each of said sample portions with regard to a diiferent enzyme constituent thereof.

The nature of the particular rate reaction of interest may, of course, require the use of detector means other and different than colorimeter means in the analysis results detector means 30. Thus, for example, said detector means may take the form of a fluorometer. Too, an AC drive motor may, of course, be substituted for the disclosed DC pump drive motor 60.

With regard to the use of the respective air segments, and especially the respective intra sample-substrate mixture air segments, it is believed clear that the primary function thereof is a cleansing function and that the same can be eliminated. Accordingly, the term portions as used in this specification is by no means intended to be limited in meaning to different parts of a Sample-substrate mixture as are in fact physically separated by a separating fluid in the nature of said intra sample-substrate mixture air segments, but rather, is intended to define different pai ts of a said sample-substrate mixture as may or may not be physically separated. Further, and with regard to the recorder means 32, it is believed clear that the same may further include digital read-out and/or print-out means which could be operated concomitantly with the strip chart recorder through the use of appropriate circuit and switching components in the nature of average slope detector means which would, of course, function to convert the analog output as applied from the colorimeter 124 on line 131 into suitable digital form for application to said digital read/out and/ or print-out means.

Also, and although disclosed herein by way of example as operating through substantially hyperbolic variation in the flow rate of the respective blood sample-substrate mixture portions through the incubation coil 78, it is believed clear that said flow rate may be varied in a manner other and dilferent than substantially hyperbolically.

From all of the foregoing, it is believed clear that the method and apparatus of the invention are particularly adaptable for incorporation in automated blood sample analysis equipment of the nature disclosed in US. Pat. 3,134,263 issued May 26, 1964 to Edward B. M. DeJong and assigned to the assignee hereof, and/or US. Pat. 3,241,432, issued Mar. 22, 1966 to Leonard T. Skeggs, et al. and assigned to the assignee hereof.

While I have shown and described the preferred embodiment of my invention, it will be understood that the invention may be embodied otherwise than as herein specificially illustrated or described, and that certain changes in the form and arrangement of parts and in the specific manner of practicing the invention may be made without departing from the underlying idea or principles of this invention within the scope of the appended claims.

What is claimed is:

1. A method of continuous-flow analysis for a constituent of interest of a plurality of different liquid samples seriatum comprising:

flowing a stream of a fluid reagent to react with each sample into the inlet of a first conduit portion having an outlet, for discharge of said reagent, coupled to an inlet of a second conduit portion, which second conduit portion comprises a reaction chamber having an outlet;

flowing each liquid sample seriatum in a stream into an inlet of said reaction chamber for treatment by exposure to said reagent therein over a period of time as the combined sample-reagent stream flows toward said outlet of said reaction chamber, the samples being maintained substantially separate and apart from one another in the last-mentioned stream; varying the rate of flow of each such treated sample through said chamber, each such variation being the same for each such treated sample and over the same time period, so that a portion of the sample has a 13 longer residence time in said chamber than another portion of the same sample; and

analyzing the quantities of the reaction product of each such treated sample over a period of time including said longer and shorter residence times, as a measurement of the quantity of said constituent of interest in each sample.

2. The method as defined in claim 1, wherein the variation of the rate of flow of each said treated sample through said reaction chamber is a hyperbolic variation, said reaction chamber comprising an incubator.

3. The method as defined in claim 1, wherein the variation of the rate of flow of each said treated sample through said reaction chamber is a hyperbolic variation, said reaction chamber comprising an incubator, each such treated sample being pumped through said incubator.

4. The method as defined in claim 1, wherein said sample is a blood sample, said constituent is an enzyme, said reagent is an appropriate substrate, and said analysis is of the optical density of the sample-substrate mixture.

5. The method as defined in claim 1, further comprising the step of, adding a reagent to said treated and reacted sample portions to effect a secondary reaction thereof.

6. The method as defined in claim 5, wherein said sec ondary reaction proceeds for substantially the same period for each of said treated sample portions.

7. The method as defined in claim 5, wherein the addition of the reagent for the secondary reaction is a colorproducing reagent, the last-mentioned reagent addition is elfected by the sampling of said sample portions at substantially constant flow rate, and the addition of said color-producing reagent is in substantially constant, predetermined proportion.

8. Apparatus of the continuous-flow type for analysis of a constituent of interest of a plurality of different liquid samples seriatum comprising: a source of discrete liquid samples; a reagent source; a reaction chamber having inlet means and having outlet means; a first conduit having a sample inlet and having an outlet connected to said inlet means of said reaction chamber; a second conduit having a reagent inlet and having an outlet coupled to said inlet means of said reaction chamber; means for flowing said sample liquid and said reagent in the respective first and second conduits through said inlet means for treatment of each liquid sample by exposure tosaid reagent Within said reaction chamber over a period of time as the combined sample-reagent stream flows toward said outlet of said reaction chamber, the samples being maintained substantially separate and apart from one another in the last-mentioned stream; means for varying the rate of flow of each such treated sample through said chamber, each such variation being the same for each such treated sample and over the same time period, so that a portion of the sample has a longer residence time in said chamber than another portion of the same sample; and analysis means coupled to said outlet means of said reaction chamber for analyzing the quantities of the reaction product of each such treated sample over a period of time including said longer and shorter residence times, as a measurement of the quantity of said constituent of interest in the sample.

9. Apparatus as defined in claim 8 wherein: said means for flowing said liquid sample and said reagent in the respective first and second conduits and said means for varying the rate of flow of each of said treated samples through said chamber comprises pump means, and said reaction chamber comprises an incubator.

10. Apparatus as defined in claim 8, wherein: said means for varying the rate of flow of each of said treated samples through said chamber comprises means to vary the last-mentioned flow hyperbolically.

11. Apparatus as defined in claim 9, wherein: said pump means is driven at a relatively fast speed and at a relatively low speed to ,vary the flow of each of said treated samples through said chamber.

12. Apparatus as defined in claim 11, wherein: said pump speed is varied hyperbolically during the flow of each of said treated samples through said chamber.

13. Apparatus as defined in claim 8, further comprising a third conduit having an inlet coupled to said outlet means of said chamber to receive from said chamber the reaction products thereof, said third conduit having an outlet coupled to said analysis means, and means to flow a reagent into said third conduit to etfect a secondary reaction therein.

14. Apparatus as defined in claim 13, wherein: said reagent is a color-producing reagent flowing in substantially constant predetermined proportion, and said analysis means comprises a colorimeter.

References Cited UNITED STATES PATENTS 3,437,447 4/1969 Harmon 195-103.5 R 3,526,480 9/1970 Findl et a1 23253 3,542,515 11/1970 Scott l103.5 R 3,241,432 3/1966 Skeggs et a1. 23253 R ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 

